Investigating lysosomal dysfunction in Fabry disease using induced pluripotent stem cell-derived podocytes

Aims: This study used induced pluripotent stem cell-derived podocytes from a Fabry disease (FD) patient carrying the p.Met284Thr pathogenic variant as an in vitro model to investigate lysosomal abnormalities driving cell pathology. Proteomic analysis was used to assess changes in lysosomal protein abundance in FD podocytes compared to controls. Additionally, temporal changes in lysosome number in FD podocytes were analyzed using automated live-cell imaging. Methods: Label-free mass spectrometry proteomics was performed on FD podocytes at day 10 of differentiation compared to controls. For live-cell imaging, cultured podocytes were transfected with CellLight Lysosomes-GFP and Plasma Membrane-CFP, and then visualized and quantified on days 10 and 20 post-differentiation using a Perkin Elmer Phenix High Content Screening Microscope. Results: Proteomic analysis showed dysregulation of glycosphingolipid metabolism proteins, including decreased galactosidase alpha (GLA; P < 0.01) and increased galactosylceramidase and glucosylceramidase (P < 0.01) in FD podocytes. Lysosomal proteins were enriched, with a significant increase in cathepsin B (P < 0.001) and a decrease in lipase A (P < 0.01). Furthermore, the dysregulation of proteins involved in cell cycle regulation and growth signaling pathways, such as polo-like kinase 1 (PLK1; P < 0.0001) and proto-oncogene tyrosine-protein kinase Src (SRC; P < 0.01), suggested broader impacts on cellular processes. Temporal live-cell imaging revealed a significant increase in lysosome number in day 20 FD podocytes compared to day 10 FD podocytes and controls (P < 0.01). Conclusions: These findings collectively suggest that FD podocytes undergo progressive lysosomal impairment, which may contribute to cellular dysfunction and disease progression. These proof-of-concept findings lay a foundation for future research on targeted FD therapies using high-throughput screening and advanced analytical techniques.